Supports and methods for promoting integration of cartilage tissue explants

ABSTRACT

The present disclosure provides tissue supports and methods for preparing a cartilage composition for repairing cartilage defects, which is prepared by expanding and integrating small cartilage tissue pieces derived from donor or engineered tissue. The methods and supports described herein promote cell migration and integration of neighboring tissue pieces in culture to form the cartilage composition. Methods of cartilage repair using the cartilage composition are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No. 61/740,787, filed Dec. 21, 2012, the entire disclosure of which is herein incorporated by reference.

TECHNICAL FIELD

The present disclosure relates generally to methods and compositions for expanding cartilage tissue explants in vitro, and in particular to a tissue support for promoting expansion and integration of cartilage tissue particles or pieces, and related methods.

BACKGROUND OF THE INVENTION

Injury and degeneration of cartilage tissue is a major clinical challenge for several reasons. In the epidemiological sense, arthritis and other degenerative joint diseases afflict a large proportion of aging populations, which are growing at high rates in most developed nations. In the clinical sense, healing of cartilage tissue is compromised by a lack of direct blood supply. When cartilage tissue alone is damaged, i.e., in the case of a chondral lesion, local chondrocytes can only achieve limited repair. A full-thickness articular cartilage injury, or osteochondral lesion, will elicit a complete inflammatory response, but results in poor tissue reformation. As a result, a surgical approach to repair and prevention of further injury can be the only viable option. Total artificial joints have been developed and used as replacements for many years with reasonable success. Total joint replacement is nevertheless costly, invasive, carries certain risks such as blood clots, blood loss and infection, and may not provide complete restoration of function. Additionally, although significant advances have been made over the last few decades in designing robust artificial joints, they do wear out. Total joint replacement in patients younger than about 60 must be carefully considered, given the risk of the artificial joint wearing out.

Tissue engineering provides an alternative approach to joint repair. Engineered tissue, including cartilage tissue, can now be prepared in vitro and then implanted in an afflicted joint to replace damaged cartilage. The technical challenge has been how to engineer a tissue that has the biomechanical properties native to cartilage, and is also biocompatible. Various approaches have been tried with differing levels of success. One approach is to obtain cells from an acceptable donor source, and seed the cells onto some sort of scaffold that provides needed mechanical support, and then maintain the arrangement in culture with appropriate nutrients and growth factors with the expectation that the seeded cells will mature, or differentiate and mature, to the desired chondrocyte phenotype. While this approach generally holds promise, multiple technical obstacles remain, arising primarily from the difficulty in finding a suitably strong biocompatible material that also promotes chondrocyte differentiation, proliferation, phenotype retention and ability of chondrocytes to produce appropriate levels of cartilage-specific glycosoaminoglycans. Certain naturally-occurring and synthetic biopolymers have been investigated for such applications, with varying degrees of success.

While significant progress has been made in successfully engineering small amounts of certain types of cartilage, many substantial barriers remain. In particular, engineered cartilage tissue that is sufficiently robust to apply to weight-bearing joints, rather than merely to cosmetic applications, remains a continuing objective. For true functionality within a joint, the resulting tissue must demonstrate the cellular characteristics and architecture of native cartilage, while commercial viability requires that the tissue be readily generated from the relatively small amounts of source tissue that is reasonably available. The field therefore continues to search for improved methods for promoting and sustaining cartilage tissue expansion from small initial amounts of donor tissue.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a support for preparing a cartilage composition from a plurality of cartilage tissue pieces, the support comprising a biocompatible material having a surface defining a plurality of tissue anchors separated by a distance sufficient to secure the plurality of tissue pieces to the support at an inter-piece distance of 1 mm or less. Each tissue piece has a starting volume less than 1 mm³. The biocompatible material may comprise a solid material, such as for example at least one trabecular metal, or a semi-solid material such as for example a gel. The tissue anchors comprise a plurality of surface features such as pins, barbs, ridges, hooks, posts, recesses and/or apertures in the biocompatible material. The support may have a plurality of cartilage tissue pieces coupled to its surface, wherein each cartilage tissue piece is coupled to a tissue anchor on the support so that the tissue pieces are separated by an inter-piece distance of 1 mm or less. A cartilage adhesive is optionally applied to the surface defining the plurality of tissue anchors. The cartilage adhesive may be applied for example to a plurality of predetermined locations on the surface of tissue anchors, in such manner as to confine the cartilage adhesive to the predetermined locations.

Also provided is a kit including any support as described herein, and a first container holding a plurality of cartilage tissue pieces. The kit may further comprise instructions for securing the plurality of cartilage tissue pieces to the tissue anchors on the support. The kit may further comprise a cartilage adhesive, which may be supplied for example in a separate container together with instructions for applying the cartilage adhesive to the surface defining the plurality of tissue anchors on the support. Alternatively, the cartilage adhesive may be applied to the surface defining the plurality of tissue anchors on the support. The cartilage adhesive may be applied for example to a plurality of predetermined locations on the surface of tissue anchors such that the cartilage adhesive is confined to the predetermined locations.

In another aspect, the present disclosure provides a tissue culture system for preparing a cartilage composition for repair of a cartilage tissue defect, the tissue culture system including: a support including a biocompatible material having a surface defining a plurality of tissue anchors separated by a distance sufficient to secure a plurality of tissue pieces to the solid support at an inter-piece distance of 1 mm or less; and a plurality of cartilage tissue pieces, each cartilage tissue piece secured to a tissue anchor. In the tissue culture system, each cartilage tissue piece may have a volume of less than 1 mm³. The tissue culture system may comprise any support as described herein. The cartilage tissue pieces may comprise for example cartilage tissue obtained from a donor, which may be from a juvenile human donor. The cartilage tissue pieces may comprise engineered cartilage tissue.

In another aspect, the present disclosure provides a method for preparing a cartilage composition for repair of a cartilage tissue defect, including: a) dividing cartilage tissue into a plurality of tissue pieces, each portion having an initial volume of less than 1 mm³, and b) maintaining the tissue pieces in a culture medium for a time and under conditions sufficient for each tissue piece to attain an expanded volume of at least 1 mm³, wherein the cell culture conditions comprise securing each tissue piece to a support, wherein the support includes a biocompatible material having a surface defining a plurality of tissue anchors separated by a distance sufficient to secure the plurality of tissue pieces to the solid support at an inter-piece distance of 1 mm or less. In the method, the support may be any of those as described herein. The cartilage tissue pieces may comprise for example cartilage tissue obtained from a donor, which may be from a juvenile human donor. The cartilage tissue pieces may comprise engineered cartilage tissue.

In another aspect, the present disclosure provides a method for repair of a cartilage defect, including: a) dividing cartilage tissue into a plurality of tissue pieces, each portion having an initial volume of less than 1 mm³, and b) maintaining the tissue pieces in a culture medium for a time and under conditions sufficient for each tissue piece to attain an expanded volume of at least 1 mm³, wherein the cell culture conditions comprise securing each tissue piece to a support, wherein the support includes a biocompatible material having a surface defining a plurality of tissue anchors separated by a distance sufficient to secure the plurality of tissue pieces to the solid support at an inter-piece distance of 1 mm or less; c) removing the cartilage composition from the support; and d) implanting the cartilage composition into the cartilage defect. In the method, the support may be any of those as described herein. The cartilage tissue pieces may comprise for example cartilage tissue obtained from a donor, which may be from a juvenile human donor. The cartilage tissue pieces may comprise engineered cartilage tissue. The method may further comprise maintaining the tissue pieces in the culture medium for a time and under conditions sufficient for at least a first expanded tissue piece to attain an expanded volume sufficient for the first expanded tissue piece to contact at least a second expanded tissue piece. The culture conditions may comprise a culture medium including at least one additive from a class of agents selected from the group consisting of: chondrocytes, progenitor cells, stem cells, hormones, growth factors and cytokines.

In another aspect, the present disclosure provides a method for repair of a cartilage defect, including: a) dividing cartilage tissue into a plurality of tissue pieces, each portion having an initial volume of less than 1 mm³; b) maintaining the tissue pieces in a culture medium for a time and under conditions sufficient for each tissue piece to attain an expanded volume of at least 1 mm³, wherein the cell culture conditions comprise securing each tissue piece to a support, wherein the support includes a biocompatible material having a surface defining a plurality of tissue anchors separated by a distance sufficient to secure the plurality of tissue pieces to the solid support at an inter-piece distance of 1 mm or less; and c) implanting and fixing the cartilage composition together with the solid support into the cartilage defect. In the method, the support may be any of those as described herein. The cartilage tissue pieces may comprise for example cartilage tissue obtained from a donor, which may be from a juvenile human donor. The cartilage tissue pieces may comprise engineered cartilage tissue. The method may further comprise maintaining the tissue pieces in the culture medium for a time and under conditions sufficient for at least a first expanded tissue piece to attain an expanded volume sufficient for the first expanded tissue piece to contact at least a second expanded tissue piece. The culture conditions may comprise a culture medium including at least one additive from a class of agents selected from the group consisting of: chondrocytes, progenitor cells, stem cells, hormones, growth factors and cytokines.

In yet another aspect, the present disclosure provides a method for preparing a cartilage composition for repair of a cartilage tissue defect, including: a) dividing an amount of cartilage tissue into a plurality of tissue pieces defining a population, each portion having an initial volume of less than 1 mm³, and b) maintaining the tissue pieces in a culture medium for a time and under conditions sufficient for each tissue piece to expand so that the average volume of tissue pieces in the population is at least 1 mm³. In the method, the culture medium may comprise any of the additives as described herein. The cartilage tissue pieces may comprise for example cartilage tissue obtained from a donor, which may be from a juvenile human donor. The cartilage tissue pieces may comprise engineered cartilage tissue may be any of those as described herein. The method may further comprise maintaining the tissue pieces in the culture medium for a time and under conditions sufficient for each tissue piece to expand so that the average volume of tissue pieces in the population is at least 1.5 mm³. Alternatively, the tissue pieces may be maintained in the culture medium for a time and under conditions sufficient for each tissue piece to expand so that the average volume of tissue pieces in the population is at least 2.0 mm³. In the method, each tissue piece may be positioned on a culture surface at an inter-piece distance of 1 mm or less. The tissue piece may be secured to the culture surface at an inter-piece distance of 1 mm or less as it is positioned. The cartilage adhesive is optionally applied to an interface between each tissue piece and the culture surface.

The cartilage adhesive may be applied for example to a plurality of predetermined locations on the surface, in such manner as to confine the cartilage adhesive to the predetermined locations. The method may further comprise maintaining the tissue pieces in the culture medium for a time and under conditions sufficient for at least a first expanded tissue piece to attain an expanded volume sufficient for the first expanded tissue piece to contact at least a second expanded tissue piece. The culture conditions may comprise a culture medium including at least one additive from a class of agents selected from the group consisting of: chondrocytes, progenitor cells, stem cells, hormones, growth factors and cytokines.

In still another aspect, the present disclosure provides a cartilage composition for repair of a cartilage tissue defect, wherein the cartilage composition includes a plurality of expanded cartilage tissue pieces defining a population, wherein each tissue piece in the population is expanded from an initial volume of less than 1 mm³ to an expanded volume such that the population of expanded cartilage tissue pieces has an average expanded volume of at least 1 mm³. The average volume of expanded cartilage tissue pieces in the population is for example at least 1.5 mm³. Alternatively, the average volume of expanded cartilage tissue pieces in the population is at least 2.0 mm³. In the cartilage composition, each expanded tissue piece in the population contacts at least one other expanded tissue piece. The cartilage tissue pieces may comprise for example cartilage tissue obtained from a donor, which may be from a juvenile human donor. The cartilage composition may further comprise a culture medium including at least one additive from a class of agents selected from the group consisting of: chondrocytes, progenitor cells, stem cells, hormones, growth factors and cytokines. In the provided cartilage composition, the expanded cartilage tissue pieces are obtained by a) dividing an initial amount of cartilage tissue into a plurality of cartilage tissue pieces defining a population, each portion having an initial volume of less than 1 mm³, and b) and maintaining the cartilage tissue pieces in a culture medium for a time and under conditions sufficient for each tissue piece to expand so that the average volume of tissue pieces in the population is at least 1 mm³. For the cartilage composition, the step of obtaining the expanded tissue pieces further comprise before step (b), the step of positioning each tissue piece on a culture surface at an inter-piece distance of 1 mm or less. As a result, the present invention also provides use of the cartilage composition as disclosed herein to repair a cartilage defect in a subject in need thereof.

In yet another aspect, the present disclosure provides a method for repair of a cartilage defect, including: implanting and fixing into the cartilage defect a cartilage composition including a plurality of expanded cartilage tissue pieces defining a population, wherein each tissue piece in the population was expanded from an initial volume of less than 1 mm³ to an expanded volume such that the population of expanded cartilage tissue pieces has an average expanded volume of at least 1 mm³. In the method, the average volume of expanded cartilage tissue pieces in the population is at least 1.5 mm³. Alternatively, the average volume of expanded cartilage tissue pieces in the population is at least 2.0 mm³. In the population of expanded cartilage tissue pieces, at least a first expanded tissue piece contacts at least a second expanded tissue piece. In one example, each expanded cartilage tissue piece in the population contacts at least one other expanded tissue piece. The cartilage tissue pieces may comprise for example cartilage tissue obtained from a donor, which may be from a juvenile human donor. The cartilage tissue pieces may comprise engineered cartilage tissue may be any of those as described herein. The cartilage composition in the method may further comprise a culture medium including at least one additive from a class of agents selected from the group consisting of: chondrocytes, progenitor cells, stem cells, hormones, growth factors and cytokines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective, top view of a first exemplary tissue support with enlarged view of tissue anchors in form of small pins projecting from the upper surface of the tissue support.

FIG. 2 is a cross-sectional view of a second exemplary tissue support, showing tissue anchors in the form of recessions on the upper surface of the tissue support.

FIG. 3 is a cross-sectional view of the first exemplary tissue support of FIG. 1, showing small tissue pieces placed on the pins.

FIG. 4 is a cross-sectional view of the second exemplary tissue support of FIG. 2, showing small tissue pieces placed in the recessions.

FIG. 5 is a series of photomicrographs showing expansion of calf and human juvenile cartilage explants in culture.

FIG. 6 is a series of photomicrographs showing non-uniform expansion of cultured cartilage explants.

FIG. 7 is a photomicrograph showing the structural interactions observed between cultured calf cartilage pieces at day 5 of culture (20×), when maintained at an inter-piece distance of 1 mm.

FIG. 8 is a photomicrograph showing the structural interactions observed between cultured human juvenile cartilage pieces at day 5 of culture (5×), when maintained at an inter-piece distance of 1 mm.

FIG. 9 is a photomicrograph showing results obtained with calf cartilage explants that were cultured with different inter-piece distances, showing structural integration of two neighboring pieces only when they the inter-piece distance was no greater than 1 mm.

FIG. 10 is a photomicrograph showing results obtained with calf cartilage explants during an initial culture phase in which inter-piece interactions including simultaneous cell migration into the inter-piece area and production of ECM. Newly formed ECM along with the cells embedded inside can be seen as a bridge between the two neighboring pieces which “glues” the cells together.

FIG. 11 is a series of photomicrographs showing the formation of inter-piece interactions of human juvenile cartilage explants. The cells migrated into the inter-piece area and produce ECM simultaneously. The newly formed ECM along, with the embedded cells, can be seen as a bridge between the two pieces which “glues” the cells together. Inter-piece interactions enclosed by the red circle in A (5×) are shown in B (20×). Inter-piece interactions enclosed by the red circles in C (5×) are shown in D and E (20×).

FIG. 12 is a pair of photomicrographs of calf cartilage explants showing structural inter-piece interactions over time, which became denser and stringer over time. The newly formed interactions contain both cells and ECM. A: day 24 of culture (20×), B: day 38 of culture (20×).

FIG. 13 is a series of photomicrographs of human juvenile cartilage explants showing the inter-piece interactions over time, which became denser and stronger over time. The newly formed interactions contain both cells and ECM. A: day 26 of culture (10×), B: day 64 of culture (10×). C: the newly formed inter-piece interactions on day 64 of culture (20×).

FIG. 14 is a pair of photomicrographs of calf cartilage explants showing structural inter-piece interactions over time, which became thicker and wider over time. Inter-piece interactions are highlighted by the red circle. A: day 42 of culture (5×), B: day 120 of culture (5×).

FIG. 15 is a series of photomicrographs of histologically stained cultured calf cartilage explants at 10 weeks of culture, in which newly formed inter-piece interaction areas are enclosed by the red circles. A: the two calf cartilage pieces at 10 weeks of culture before histology staining B: and C: Masson's trichrome staining, D: and E: H:-E: staining, F: Alcian blue staining and G: Safranin O Staining C: and E: show the cell migration out of the edges of the original calf cartilage pieces.

FIG. 16 is a pair of photomicrographs showing cultured cartilage pieces positioned at an inter-piece distance of greater than 1 mm were mainly surrounded by the migrated cells. A: calf cartilage explants (10×), B: human juvenile cartilage explants (10×).

FIG. 17 is a pair of photomicrographs of results after 10 weeks of culture, showing cells that migrated out of human juvenile cartilage explant proliferated and accumulated next to the original tissue pieces, where they piled up as multiple layers and reached approximately the same height of the cartilage pieces. A: 5×, B: 10×.

FIG. 18 is a series of photomicrographs showing effects of cartilage harvesting locations: A: trochlea, B: trochlea groove, C: femoral condyle, D: central tibial plateau and E: peripheral tibial plateau. Pictures were taken on day 35 of culture.

FIG. 19 is a photomicrograph showing results obtained with calf cartilage explants of different sizes.

FIG. 20 is a series of photomicrographs showing migrated cells from calf cartilage explants having different phenotypes, on TC and ULA plates. A: the bottom layers of cell attached to the bottom area of TC plates on day 35 of culture (10×). B: The initial formation of cell cluster from individual cells on ULA plates on day 35 of culture (10×). C: Multiple layers of cells formed on the bottom of TC plates on day 120 of culture. The top layer of cells showed un-stretched round shape (10×). D: More and bigger cell clusters were formed on ULA plates on day 120 of culture, the majority of cells inside the cluster showed round shape (20×).

DETAILED DESCRIPTION

The present disclosure is based in part on the surprising discovery that small cartilage tissue pieces, when maintained in vitro under culture conditions, will expand and structurally integrate with neighboring tissue pieces when maintained at an inter-piece distance of 1 mm or less than 1 mm. By “inter-piece distance” is meant that distance from an outside surface of one tissue piece to the closest outside surface of a second tissue piece adjacent to the first tissue piece. Exemplary tissue pieces have a volume of less than about 1 mm³, and may be of many possible shapes, e.g., cubes, cylinders, ovoids, and the like, which may be produced according to the method of preparing the pieces from a tissue source. An outside surface of a tissue piece may therefore be substantially linear or arcuate, depending on the shape of the tissue piece. For example, tissue pieces may be substantially cuboid, with substantially linear outer surfaces, and have a volume of less than about 1 mm³, e.g., dimensions of less than approximately 1 mm on each side. It has now been found that two neighboring cartilage pieces when placed no more than 1 mm apart, form new tissue through the activity of cells that migrate from each piece and interact to form a structurally integrated larger piece of tissue over time. In marked and surprising contrast, neighboring such tissue pieces that are placed more than 1 mm apart do not expand and integrate in the same way. Importantly, it has further now been demonstrated that when placed and maintained while in culture as described, cells from the initial tissue pieces will migrate into the area between two cartilage pieces and produce extracellular matrix (ECM) simultaneously. The newly formed ECM, along with cells embedded therein, forms new inter-piece tissue that binds neighboring pieces together. Additionally, more cells migrate over time onto the inter-piece tissue, and cells already embedded inside the ECM also continue to proliferate. As a result, the ECM becomes denser, and the volume of the inter-piece tissue, which consists of multiple layers of ECM and cells embedded therein, increases. The increase in tissue volume is reflected in an increase in height, width and/or length of the inter-piece tissue, which also becomes structurally integrated with the neighboring tissue pieces. Particularly surprising is the finding that the thus newly formed inter-piece tissue exhibits structure that is comparable to that of the original cartilage tissue, based on gross and histological evaluation. The expanded tissue thus provides a new source for a cartilage composition that can be used for cartilage repair.

A. DEFINITIONS

Section headings as used in this section and the entire disclosure herein are not intended to be limiting. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.

As used herein, the term “about” refers to approximately a +/−10% variation from the stated value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

As used herein, the term “biocompatible material” or biomaterial refers to a synthetic or natural material that can be used to replace part of a living system or to function when in close contact with living tissue.

As used herein, the term “cartilage” refers to an avascular tissue, having chondrocytes encapsulated within an extracellular matrix. As used herein, cartilage tissue may be donor cartilage tissue or engineered cartilage tissue. “Cartilage,” as used herein, encompasses articular cartilage, hyaline cartilage, neocartilage (Adkisson, H. D. et al., Clin. Orthop. 391S: S280-S294, 2001; and U.S. Pat. Nos. 6,235,316 and 6,645,316), devitalized cartilage, auricular cartilage, cartilage including genetically modified chondrocytes, cartilage from an autogenous source, cartilage from an allogenic source, cartilage from a xenogeneic source, juvenile cartilage, or a combination thereof. In some configurations, cartilage can also comprise chondrocytes differentiated from precursor cells such as mesenchymal stem cells.

As used herein, the term “cartilage adhesive” refers to molecular species or mixtures of species which promote adhesion of cartilage tissue or chondrocytes of the cartilage tissue to a surface, by acting as a binding agent (e.g., a glue) and/or by promoting adhesion-forming activity of cells. A cartilage adhesive can be used as a binding agent (e.g., a glue) at the interface between cartilage or chondrocytes thereof and a surface.

The term “cartilage defect” refers to a structural and/or biological imperfection in cartilage tissue such as but not limited to a break, tear, void or other disintegration of the tissue, which is caused by a disease, injury or condition and which can benefit from cartilage repair, replacement, or augmentation, such as, in non-limiting example, athletic injury, traumatic injury, congenital disorders, osteoarthritis and joint degeneration from aging.

As used herein, the term “chondrogenic cell” as used herein refers to chondrogenic progenitor cells (CPCs), the further differentiation of which results in chondrocytes. Chondrogenic cells can come from a variety of sources. Generally, the chondrogenic cells can be isolated from an articular cartilage or a fibrocartilage. Chondrogenic cells from a fibrocartilage can be obtained from costal, nasal, auricular, tracheal, epiglottic, thyroid, arytenoid and cricoid cartilages. Alternatively, cells from fibrocartilage can be obtained from tendon, ligament, meniscus and intervertebral disc. Depending on the context, chondrogenic cells may refer to partially differentiated progenitor cells destined to be chondrocytes or chondrogenic stem cells that are more primitive. The chondrogenic stem cells can be derived from a tissue such as placenta, umbilical cord, bone marrow, skin, muscle, fat, periosteum, and perichondrium.

The term “subject” as used herein refers to a mammal, which may be a human or a non-human mammal such as but not limited to a horse, a dog, a cat, a non-human primate such as a monkey or ape, a rabbit, a rat, a mouse, or a pig.

The term “trabecular metal,” as used herein, encompasses any biocompatible metal or metal composite having interconnecting pores and at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 85% porosity by volume. Exemplary such metals have pores of about 200 μm-700 μm in diameter, and preferably 400 μm-600 μm in diameter. Descriptions of trabecular metal, as well as various methods of making trabecular metal of various pore sizes are known in the art. An exemplary such material is a tantalum composite sold as Trabecular Metal® by Zimmer, Inc.

B. TISSUE SUPPORTS

In one aspect, the present disclosure provides a support for preparing a cartilage composition from a plurality of cartilage tissue pieces, the support including a biocompatible material having a surface defining a plurality of tissue anchors separated by a distance sufficient to secure the plurality of tissue pieces to the support at an inter-piece distance of 1 mm or less. Tissue pieces of varying initial volumes may be used. A tissue piece may for example have a starting volume of about 1 mm³, or less than about 1 mm³, or more than about 1 mm³. For example, a population of tissue pieces having an initial average volume of less than 1 mm³, maintained at an inter-portion distance of 1 mm or less, can expand as described herein to attain an average volume of at least 1.5 mm³ or 2.0 mm³ and thereby integrate with surrounding pieces. It should nevertheless be appreciated that if the tissue pieces are cultured under more favorable expansion conditions, for example by the addition of growth factors to the culture system, the tissue pieces may attain a greater volume within a given period of time, and thereby integrate two neighboring pieces that are maintained at a distance of more than 1 mm apart.

Similarly, with respect to the initial volume of the tissue pieces, although an exemplary tissue piece size is about 1 mm³, or less than about 1 mm³, the initial tissue size (volume) could be larger than about 1 mm³ and still suitably expand as described herein.

Tissue pieces may vary in shape and may be for example substantially spherioid, cuboid, cylindrical, or ovoid. It should be understood that the shape of the tissue pieces will depend in part on the method used to prepare tissue pieces from the donor tissue. A non-limiting exemplary tissue piece is substantially cuboid and has a starting volume less than about 1 mm³, e.g., dimensions of less than approximately 1 mm on each side. The biocompatible material may comprise a solid material, such as for example at least one trabecular metal, or a semi-solid material such as for example a gel.

The tissue anchors comprise a plurality of surface features such as pins, barbs, ridges, hooks, posts, recesses and/or apertures in the biocompatible material. The support may have a plurality of cartilage tissue pieces coupled to its surface, wherein each cartilage tissue piece is coupled to a tissue anchor on the support so that the tissue pieces are separated by an inter-piece distance of 1 mm or less. A cartilage adhesive is optionally applied to the surface defining the plurality of tissue anchors. The cartilage adhesive may be applied for example to a plurality of predetermined locations on the surface of tissue anchors, in such manner as to confine the cartilage adhesive to the predetermined locations. Alternatively, the tissue anchors may be provided solely as adhesive spots in predetermined locations on an otherwise featureless surface of the support.

Accordingly, the present disclosure encompasses tissue supports for maintaining tissue pieces at an inter-piece distance of 1 mm or less. As shown in FIG. 1 for example, a tissue support 100 is generally made of a biocompatible material 102 having a surface 104 defining multiple tissue anchors 106 for securing tissue pieces to support 100. The tissue anchors may take the form of any of a number of suitable surface projections from surface 104. As shown in FIG. 1, for example, each tissue anchor is a pin 108. Other projecting forms suitable for securing tissue pieces can be used, such as but not limited to barbs, ridges, hooks, or straight or curved posts. Alternatively, the tissue anchors may generally take the form of depressions or recesses 110 in surface 104, as shown in FIG. 2, or they may consist of apertures through biocompatible material 102. Such depressions, recesses or apertures through biocompatible material 102 may for example be configured with a shape and/or dimensions approximately matched to the shape and/or dimensions of the tissue pieces. For example, apertures for receiving substantially spherical tissue pieces may be configured as round holes through biocompatible material 102, having a diameter approximating the average diameter of the tissue pieces. Alternatively, the tissue anchors may consist solely of defined adhesive locations (not shown) on surface 104, wherein the adhesive locations are created by applying a cartilage adhesive to defined locations, e.g., as “dots” or “spots” of adhesive, on surface 104, which is otherwise free of physical anchors such as projecting anchors, or depressions or apertures.

The multiple tissue anchors are distributed substantially uniformly across surface 104 in any configuration such that each anchor is positioned at a maximum distance of about 1 mm to about 2 mm from at least one neighboring tissue anchor. The distribution of tissue anchors across surface 104 may be according to any regular or irregular pattern, provided that each anchor is no more than a maximum distance of about 1 mm to about 2 mm from at least one neighboring anchor, so that each tissue piece is able to expand and integrate with at least one neighboring piece. It will be appreciated that any distribution pattern that maximizes packing of the initial tissue pieces across surface 104 within the specified maximum distance, and thus any regular, geometric distribution pattern of the tissue anchors, will be preferred.

It should further be appreciated that the tissue anchor dimensions may suitably vary depending on the size and/or density of the tissue piece(s), and on the shape of the anchor. Tissue anchors should be sized and shaped such that the act of applying and securing a tissue piece to an anchor will not obliterate most or all of the tissue piece. For example, a tissue piece having a generally cuboid shape of about 1 mm on a side, or a thinner tissue piece of about 1 mm in length and 1 mm in width, may be anchored to the support surface using an anchor configured as a straight pin, with a largest diameter of about 50-500 microns. Anchors for tissue pieces larger than about 1 mm on a side may be appropriately sized to have a larger diameter or other cross-sectional area.

It should further be appreciated that the tissue support shape may be varied to approximate the shape of an anatomical target, such that the tissue generated on the support in vitro will approximate the target anatomical shape. For example, a support may have an arcuate shape such that the resulting tissue is appropriately shaped to be implanted at the acetabulum, head of the femur or other curved anatomic surface. A support may have a generally pyramidal shape for implantation at a talar dome lesion or fracture. Other shapes suitably adapted for other anatomical targets are also contemplated.

Each tissue piece is secured to surface 104 by applying, e.g. manually, each piece at its approximate center onto a tissue anchor, as shown in FIG. 3 and FIG. 4. It should therefore be appreciated that with initial tissue particles having approximate dimensions of 1 mm on each side, using tissue anchors that are separated by a distance of about 1 mm will provide an inter-piece distance of about 0.0 mm, e.g., the initial tissue pieces are in contact or very close to contacting neighboring piece(s). Tissue anchors that are separated by a distance of about 2 mm will provide an inter-piece distance of about 1.0 mm, i.e., a distance at which the initial tissue pieces are at or close to the maximum inter-piece distance, according to the findings and methods disclosed herein. Each piece is secured in position on an anchor by piercing, lancing or hooking the piece onto a projecting anchor on surface 104, or by depositing (e.g., by manually pressing) each piece at least partially into an anchor that is a depression, recess or anchor in surface 104. To use a tissue support that has only adhesive locations as anchors, the cartilage adhesive is applied, e.g. by manually “dotting” or “spotting” the cartilage adhesive onto surface 104 in selected locations, applying a population of tissue pieces to surface 104, waiting for a period of time sufficient for a subset of the tissue pieces to bind or adhere to the selected locations, and then removing all unbound or non-adhered tissue pieces, e.g. by washing or rinsing the unbound pieces way.

A cartilage adhesive is optionally used to further secure each tissue piece in position on the tissue support, for example by the applying cartilage adhesive to an interface between each tissue piece and a tissue support surface. Depending on the configuration of the tissue support, e.g. the type of anchors being used, a cartilage adhesive may be applied to tissue anchor surfaces, or to inter-anchor regions of the tissue support, or to both. Use of a cartilage adhesive may be desired for example when the support has anchors that do not otherwise secure the tissue pieces to the support through physical means such as piercing or hooking, e.g. depressions or apertures. For avoidance of doubt, cartilage adhesive may be used with any of the anchor forms described herein. A cartilage adhesive may be any biologically compatible composition capable of adhering to both biocompatible material 102 and the tissue pieces. For example, a cartilage adhesive may be, but is not limited to, a fibrin-based adhesive, a collagen-based adhesive or a combination thereof. A cartilage adhesive can comprise for example tissue trans-glutaminase, hyaluronic acid, collagen type I, collagen type II, a chemically cross-linked collagen, fibrin, albumin, gelatin, elastin, silk, demineralized bone matrix, polyethylene oxide, polyethylene glycol, polyvinyl alcohol, polypropylene fumarate or a combination thereof as described elsewhere, or a hydrogel. (See, e.g., Jurgensen et al., J. Bone and Joint Surg. 79A: 185-193, 1997; U.S. Pat. No. 6,893,466 to Trieu; U.S. Pat. No. 6,835,277 to Goldberg et al). In various aspects, a vertebrate-derived component of a cartilage adhesive, such as tissue trans-glutaminase, hyaluronic acid, collagen type I, collagen type II, fibrin, albumin, gelatin, or elastin, or demineralized bone matrix, can be autologous, allogeneic, or xenogeneic to a mammalian recipient of an implant, such as a human patient in need of treatment. Furthermore, a protein or polypeptide component of a cartilage adhesive such as tissue trans-glutaminase, hyaluronic acid, collagen type I, collagen type II, fibrin, albumin, gelatin, or elastin, can be obtained from a naturally-occurring source such as an animal or human donor, or can be produced using molecular biological methods well known to skilled artisans, such as expression of a gene or cDNA encoding the protein in transformed or transfected cells (see, e.g., Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). A cartilage adhesive may also comprise one or more cell-attachment factors, such as collagen I or collagen IV, fibronectin, laminin, or poly-lysine, or another commercially available cell attachment factor, inasmuch as chondrocytes are exposed at a surface of a tissue piece.

The cartilage adhesive may be applied for example to a plurality of predetermined locations on the surface of tissue anchors, in such manner as to confine the cartilage adhesive to the predetermined locations. The cartilage adhesive may be applied for example directly to any projecting anchor such as a pin, hook or barb, to complement the physical coupling of each tissue piece to such an anchor. Alternatively, the cartilage adhesive may be applied to the surface of the support surrounding an anchor that is a depression, recess or aperture through the support, or to the depression or recess itself, or to the internal walls of an aperture, or any combination thereof.

Biocompatible material 102 may comprise any of a number of materials suitable for use in tissue culture and may comprise a material or materials that enhance cell migration, cell attachment and the formation of extracellular matrix of the cells. Such materials include for example a metal, either a single metal or a combination of metals. Accordingly, titanium, cobalt, chromium, platinum, iridium, niobium or tantalum may be used, or any alloy thereof, and also stainless steel. Any metal may be a trabecular metal. Ceramics may be used, such as but not limited to calcium hydroxylapatite (dense or porous), bioactive glass (e.g., Bioglass®, Ceravital®), bioactive glass-ceramics (A/W glass-ceramic such as Cerabone®, or dense or porous hydroxyapatite), and workable glass-ceramics and phosphates glasses such as Bioverit® I, II and III. Alternatively, suitable biocompatible materials also include biopolymers such as poly-lactic acid (PLA), poly-glycolic acid (PGA), Dacron, collagen, PET (poly-ethylene terephthalate), poly-carbonate, poly-tetrafluoroethylene, and co-polymers of PLA and PGA. Alternatively, the biocompatible material may comprise a semi-solid material such as for example a gel, such as a hydrogel, or a wax. A biocompatible material may include any combination of any of the above, for example but not limited to a layered configuration with one or more core layers of certain material(s) covered by a surface layer of a different material. Alternatively, protein layers may be attached to the surface of any material to promote cell attachment.

It will be appreciated that the tissue support, though illustrated in the accompanying drawings as a substantially flat surface with tissue anchors projecting therefrom, may alternatively be configured in any shape such as one specially adapted for implantation into a particular defect, provided only that the shape includes at least one surface capable of bearing multiple tissue anchors. It should be further recognized that certain biocompatible materials suitable as the support material and also having some amount of flexibility, such as gels, waxes and any composite material containing a gel or wax, may be especially suitable for in vitro applications.

Methods for manufacturing a tissue support as described will depend on the material(s) selected and desired shape of the support, and are well known in the art. Precision manufacturing services are provided by many contract manufacturing organizations specializing in the manufacture of medical devices which provide manufacturing services including casting, extrusion, molding, and precision machining including milling and micromachining techniques such as microfinishing (deburring and polishing).

C. METHODS

The present disclosure encompasses methods for preparing a cartilage composition by dividing cartilage tissue into a plurality of tissue pieces, each having an initial volume of less than 1 mm³, and maintaining the tissue pieces in a culture medium for a time and under conditions sufficient for each tissue piece to attain an expanded volume of at least 1 mm³ or more, e.g. at least 1.5 mm³ or at least 2 mm³. The present disclosure also provides a method for repair of a cartilage defect by implanting and fixing into the cartilage defect a cartilage composition including a plurality of expanded cartilage tissue pieces having an average expanded volume of at least 1 mm³, at least 1.5 mm³ or at least 2 mm³.

In the methods, sufficient time for each tissue piece to attain an expanded volume of at least 1 mm³ or more is at least about 4 days, and can be for example as long as 14 days. While each piece may not actually attain an expanded volume of at least 1 mm³ or more in the time period, it is contemplated that a minimum time of about 4 days under appropriate culture conditions is sufficient for at least 10%, 20%, 30%, 40%, 50%, 60, 70%, 80%, 90% or 100% of the tissue pieces to attain an expanded volume of at least 1 mm³ or more. It should be understood that due to variation among pieces including differences in the microclimate, not all pieces will expand and integrate with neighboring pieces at the same rate. Longer periods of time will result in more pieces attaining a minimum expanded volume, and also more pieces that attain an expanded volume greater the minimum expanded volume.

Tissue culture conditions suitable for expanding and maintaining cartilage tissue as described herein are well known in the art. Generally, tissue including actively dividing cells is grown and maintained at an appropriate temperature which for mammalian cells, is at or about 37° C., and using a gas mixture of 5% CO₂. To help accurately control conditions, tissue may be maintained for example in an incubator, in a growth medium. It will be appreciated that the formulation for a suitable growth medium may be varied along several factors: pH, glucose concentration, number and amount of growth factors, and presence of other nutrients. Growth factors derived from a number of sources may be used, including whole animal serum such as bovine calf serum. A suitable growth medium is, in non-limiting example, DMEM/F12 medium supplemented with fetal bovine serum (FBS). Alternatively, a serum-free medium or a chemically defined medium may be used, which may be supplemented with ascorbate and/or glutamine. A suitable such growth medium is, in non-limiting example, HL-1 serum-free medium (Lonza Walkersville, Inc., Walkersville, Md.) supplemented with at least about 2 mM or more of glutamine, and about 50 to about 100 μg/ml ascorbate. A culture medium may include at least one additive from a class of agents selected from the group consisting of: chondrocytes, progenitor cells, stem cells, hormones, growth factors and cytokines, or any composition containing such an additive or additives such as platelet rich plasma (PRP). Progenitor cells and stem cells include for example cartilage progenitor cells or bone marrow cells. Growth factors and cytokines may be any from among the many known such agents, including but not limited to ECGF (Endothelial Cell Growth Factor), VEGF 168, VEGF 145, VEGF 121, aFGF (Fibroblast Growth Factor, acidic), bFGF (Fibroblast Growth Factor, basic), EGF (epidermal growth factor), PDGF (platelet derived growth factor), FGF-10, FGF-4, FGF-5, FGF-6, FGF-8b, FGF-9, IGF (insulin-like growth factor), KGF (Keratinocyte Growth Factor), TGF-β (transforming growth factor β), collagen, hypothemycin, interleukin-8 (IL-8), and IL-13. The hormones may be chosen from hydrocortisone, insulin, triiodothyronine, thyroxine, Retinyl acetate, Activin A, (2-Hydroxypropyl)-β-cyclodextrin, Methyl-β-cyclodextrin, and Hydrocortisone 21-hemisuccinate.

The cell culture conditions may include for example maintaining the tissue pieces at an inter-piece distance of 1 mm or less. This may be accomplished for example by securing each tissue piece to a tissue support as described herein. In use, as shown in FIGS. 3 and 4, tissue support 100 has a plurality of cartilage tissue pieces coupled to its surface via the tissue anchors on support 100, such that the tissue pieces are separated by an inter-piece distance of 1 mm or less. It should be understood however that other means for maintaining the inter-piece distance are encompassed by the present disclosure.

The methods further encompass methods for repairing a cartilage defect, for example in a subject. The cartilage expansion methods as described herein may be applied for example to methods to prepare a cartilage composition in vitro for subsequent implantation into a subject for repair of a cartilage tissue defect in the subject. Alternatively, the approach can be adapted to methods to generate a cartilage composition in vivo in the subject.

A method for repair of a cartilage defect may include for example producing in vitro a cartilage composition on a support, from cartilage pieces each having an initial volume of less than 1 mm³ as described herein, by expanding the initial cartilage pieces in vitro to an expanded volume of at least 1 mm³, followed by removing the resulting cartilage composition from the support, and then implanting and fixing the cartilage composition into the cartilage defect. Alternatively, a method for repair of a cartilage defect may include producing in vitro a cartilage composition on a support as described herein, and then implanting the cartilage composition together with the solid support into the cartilage defect, and closing the surgical opening such that the tissue pieces expand in vivo to an expanded volume of at least 1 mm³ or more. It will be appreciated that the latter alternative favors the use of a tissue support that is either prepared from a flexible material or materials, or is configured in a shape that fits closely with the defect being repaired.

Alternatively, a method for preparing a cartilage composition for repair of a cartilage tissue defect may involve a population of tissue pieces, wherein a) an amount of cartilage tissue is divided into a plurality of tissue pieces defining a population, each portion having an initial volume of less than 1 mm³, and b) the tissue pieces are maintained in a culture medium for a time and under conditions sufficient for each tissue piece to expand so that the average volume of tissue pieces in the population is at least 1 mm³. The tissue pieces may be maintained for a time and under conditions sufficient for each tissue piece to expand such that the average volume of tissue pieces in the population is greater than 1 mm³, for example at least 1.5 mm³, or at least 2.0 mm³. This approach may include positioning each tissue piece on a culture surface, such as a tissue support as described herein, at an inter-piece distance of 1 mm or less before the expansion step (b). Each such tissue piece may be secured to such a support by means of an anchor or a cartilage adhesive, both as described herein, or a combination thereof.

In any of the methods, the tissue pieces, which may be a population of tissue pieces, may be maintained for a time and under conditions sufficient for at least one, or more, of the tissue pieces to expand and thereby attain an expanded volume that is sufficient for the tissue piece(s) to contact at least one neighboring tissue piece that has also expanded. In an exemplary method, a starting population of tissue pieces is maintained for a time and under conditions sufficient for a simple majority if tissue pieces in the population, or for as many as all tissue pieces in the population, or for any number of tissue pieces in between a simple majority and the entire population, to expand and thereby attain an expanded volume that is sufficient for the tissue piece(s) to contact at least one neighboring tissue.

By repair is meant a surgical repair such as an open surgical procedure (arthrotomy) or an arthroscopic procedure by which the in vitro prepared cartilage composition is implanted into the defect, with or without the tissue support. The cartilage composition, or cartilage composition together with the support, may be fixed in the defect using a mechanical fastener such as a staple, screw, pin or the like, or a tissue adhesive such as for example any adhesive described elsewhere herein.

In any of the methods, cartilage tissue pieces or pieces may, without limitation, be prepared from donor or engineered cartilage tissue such as neocartilage, or a combination thereof. Source tissue may be allogeneic to, autologous to, and/or xenogeneic to a mammalian recipient such as a human patient. Cartilage tissue pieces may comprise for example, hyaline cartilage or any tissue including chondrocytes having the potential to generate hyaline cartilage, but not necessarily organized into histologically recognizable cartilage. Such tissue includes, for example, articular joint cartilage including knee joint and hip joint cartilage, tracheal cartilage, laryngeal cartilage, costal cartilage, epiphyseal plate cartilage, and any combination thereof. It will be understood that the methods and devices can be utilized to produce cartilage composition of various sources to meet the needs for treating cartilage defects of any origin. Donor cartilage tissue may be obtained from a live or a deceased donor. Donor cartilage tissue may be obtained for example from a juvenile donor, for example a donor less than fifteen years of age, preferably fourteen years of age or younger, and more preferably two years of age or younger. Donor cartilage tissue may be obtained for example from a prenatal or neonatal donor no older than about one (1) week of age. Donor cartilage tissue may be obtained from a cadaver of an individual of juvenile age at time of death.

Tissue pieces may be prepared from any donor or engineered source tissue by dividing the tissue into cuboids each having a volume less than 1 mm³, e.g cuboids of approximately 1 mm on each side. Dividing may be accomplished for example using a microcutter as known in the art. Multiple such tissue pieces may then be placed in culture, at an edge-to-edge distance of no more than 1 mm from at least one adjacent tissue piece.

Cartilage defects that may be treated with a cartilage composition as described herein encompass any cartilage defect caused by injury or disease including any acute, partial, or full-thickness chondral injury, osteochondral injuries, and defects resulting from degenerative diseases or processes. The cartilage defect can be, for example and without limitation, the result of osteochondritis dissecans (OCD), osteoarthritis, rheumatoid arthritis, or osteonecrosis.

D. KITS AND TISSUE CULTURE SYSTEMS

Also provided is a kit including a tissue support as described herein, and a first container holding a plurality of the pre-expansion cartilage tissue pieces as also described herein. The kit may be suitably used for investigative or therapeutic purposes to prepare a cartilage composition as described herein from tissue pieces. The kit may further contain, for example, instructions for securing the plurality of cartilage tissue pieces to the tissue anchors on the support. The kit may further comprise a cartilage adhesive, which may be supplied for example in a separate container together with instructions for applying the cartilage adhesive to the surface defining the plurality of tissue anchors on the support. Alternatively, the cartilage adhesive may be applied to the surface defining the plurality of tissue anchors on the support. The cartilage adhesive may be applied for example to a plurality of predetermined locations on the surface of tissue anchors such that the cartilage adhesive is confined to the predetermined locations.

A tissue culture system for preparing a cartilage composition for repair of a cartilage tissue defect may comprise a tissue support as described herein, and a plurality of cartilage tissue pieces, each cartilage tissue piece secured to a tissue anchor as also described herein. In the tissue culture system, each cartilage tissue piece has an initial volume of less than 1 mm³, and may reach an expanded volume of at least 1 mm³, at least 1.5 mm³ or at least 2 mm³. The cartilage tissue pieces in the system may comprise cartilage tissue pieces prepared from any donor or engineered source as described herein. The tissue culture medium may further any growth medium as described herein.

E. ADAPTATIONS OF THE METHODS OF THE PRESENT DISCLOSURE

Various embodiments of the present teachings can be illustrated by the following non-limiting examples. The following examples are illustrative, and are not intended to limit the scope of the claims.

Example 1 Integration of Cultured Cartilage Explants

Cartilage explants were obtained from calf and human juvenile donor tissue and prepared using a microcutter to a substantially cuboid shape of about 1.5 mm on a side. Four 1.5 mm diameter cartilage pieces were placed on a regular tissue culture plate at varying distances relative to each other: at 0, 0.5, 1.0, 2.0 and 3.0 mm and maintained in an incubator at 37° C., 5% CO₂, in a growth medium as follows: all cultures were started with DMEM/F-12/FBS medium and switched to HL-1 serum-free medium after two weeks of culture. DMEM/F-12/FBS medium was prepared by supplementing DMEM/F12 medium with 10% (v/v) fetal bovine serum (FBS, Invitrogen) supplemented with ascorbate and L-glutamine as described above, 10,000 U/ml penicillin G, 10,000 U/ml streptomycin sulphate and 25 μg/ml amphotericin B (Invitrogen). Serum free HL-1 medium was prepared from HL-1 serum-free medium (Lonza Walkersville, Inc., Walkersville, Md.) supplemented with 1:100 diluted HL-1 supplement (Lonza Walkersille, Inc.) supplemented with ascorbate and L-glutamine as described above 10,000 U/ml penicillin G, 10,000 U/ml streptomycin sulphate and 25 μg/ml amphotericin B.

Both calf and human juvenile cartilage explants became bigger and thicker over time. On average, a calf cartilage piece with a 1.5 mm diameter and 1 mm thickness increased 0.6 mm to 1 mm in diameter, and 0.5 to 0.8 mm in thickness over eight weeks of culture (FIG. 5). The cultured cartilage explants did not expand uniformly (FIG. 6). At the surface of the explant adjacent to the surface of the culture plate (“bottom surface” of the explant), the explant expanded more rapidly than at the upper surface, resulting in an asymmetric, generally cylindrical shape with a smaller upper surface relative to a bigger bottom surface, resulting in newly formed tissue appearing as a ring-like structure surrounding the original piece. Spatial reorientation of an explant piece in culture resulted in the same effect on the former upper surface reoriented as the bottom surface. An explant piece flipped over following faster expansion of the initial bottom surface, resulted in comparably rapid expansion of the new bottom surface, so that the resulting explant ended up with two “bigger” surfaces on both sides. Spatial reorientation thus may provide a method of controlling/directing the orientation and shape of cartilage explant expansion.

Interactions among the cartilage pieces were generated as early as day 5 of calf cartilage culture (FIG. 7) or day 7 of human juvenile cartilage culture (FIG. 8). These tissue cultures demonstrated that two cartilage pieces were able to structurally integrate to each other only when they were in close proximity, 1 mm or less than 1 mm apart. This occurred in one of two ways: (a) the pieces were placed in contact with each other at the beginning, or (b) the pieces were placed no more than 1 mm apart and the newly formed tissue by the migrated cells integrated with both explants to form a larger piece of tissue over time (FIG. 9).

Initiation of Inter-Piece Interactions:

During cell migration, some cells migrated into the area between two cartilage pieces and produced extracellular matrix (ECM) simultaneously. The newly formed ECM along with the embedded cells generated the new inter-piece interactions which bind, adhere or “glue” these two pieces together (FIGS. 10 and 11). Over time, more and more cells migrated onto the bridge area between these two pieces. In addition, the cells that were already embedded inside the ECM continued to proliferated. As a result, the ECM became denser and the inter-piece interactions became thicker and wider, containing multiple layers of ECM and cells embedded therein (FIGS. 12, 13 and 14), and structurally integrated with the surrounding explanted tissue. The structure of the newly formed inter-piece interaction was found to be similar to the original cartilage tissue, based on the gross and histological observation (FIG. 15). In contrast, those cartilage explant pieces that were placed more than 1 mm apart were unable to integrate together. Instead, they were surrounded by the migrating and proliferating cells (FIG. 16). The cells accumulated next to the cartilage piece, where they piled up as multiple layers and reached almost the same height of the original piece at 10 weeks of culture (FIG. 17).

Example 2 Effect of Cartilage Harvesting Location and Implant Size

Calf cartilage explants were harvested from five different locations of knee joint: trochlea, trochlea groove, femoral condyle, central tibial plateau and peripheral tibial plateau. The pieces of calf cartilage from different sources were placed according to methods in Example 1. No significant difference was observed on cell migration, cartilage explant expansion and integration among the calf cartilage explants harvested from different locations of knee joint (FIG. 18). Further evaluation was carried out with different sizes of calf cartilage explants. Calf cartilage explants of 1.5 mm, 2 mm, 3 mm and 4 mm in diameter were prepared and cultured according to methods as otherwise described in Example 1. No significant difference was observed on cell migration, cartilage explant expansion and integration among different sizes of calf cartilage explants (FIG. 19).

While there was no significant difference between ULA (ultra low attachment) and TC (regular tissue culture) plates on cell migration, cartilage explant growth and integration, significant differences were observed between these two plates on the phenotype of the migrated cells: on TC plates, the migrated cells proliferated and attached to the bottom areas of the plates. The over confluent cells formed multiple layers. The cells on the bottom layers became stretched showing fibroblast-like phenotype, whereas the cells on the top layer still maintained their original round shape showing the typical phenotype of chondrocytes. On ULA plates, instead of attaching to the bottom of the plates, the migrated cells attached to each other and formed cell clusters. Even though some cells on the bottom of the clusters became stretched and attached to the plates, the majority of the cells in the cluster still maintain their original round shapes (FIG. 20).

One skilled in the art would readily appreciate that the methods described in the present disclosure are well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods, procedures, treatments, described herein are merely representative and exemplary, and are not intended as limitations on the scope of the invention. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the present disclosure disclosed herein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the present disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. 

1-20. (canceled)
 21. A method of making expanded cartilage tissue, the method comprising: a) placing a plurality of cartilage tissue pieces on a solid support at an inter-piece distance of 1 mm or less, wherein each cartilage tissue piece has an initial volume of less than 1 mm³; and b) culturing the plurality of cartilage tissue pieces on the solid support for a time and under conditions sufficient for each cartilage tissue piece to attain an expanded volume of at least 1 mm³.
 22. The method of claim 21, wherein the average volume of expanded cartilage tissue pieces is at least 1.5 mm³.
 23. The method of claim 21, wherein the average volume of expanded cartilage tissue pieces is at least 2.0 mm³.
 24. The method of claim 21, wherein each expanded tissue piece contacts at least one other expanded tissue piece.
 25. The method of claim 21, wherein the cartilage tissue pieces comprise donor cartilage tissue.
 26. The method of claim 25, wherein the donor cartilage tissue is allogeneic to a mammalian recipient of the plurality of expanded cartilage tissue pieces.
 27. The method of claim 21, wherein the cartilage tissue pieces comprise engineered cartilage tissue.
 28. The method of claim 21, wherein the cartilage tissue pieces comprise human juvenile cartilage tissue.
 29. The method of claim 21, wherein the culturing comprises culturing in a culture medium.
 30. The method of claim 29, wherein the culture medium is serum-free.
 31. The method of claim 29, wherein the culture medium comprises at least one additive.
 32. The method of claim 31, wherein the at least one additive comprises cells.
 33. The method of claim 32, wherein the cells are at least one of chondrocytes, progenitor cells, or stem cells.
 34. The method of claim 31, wherein the at least one additive comprises at least one bioactive agent.
 35. The method of claim 34, wherein the at least one bioactive agent is at least one of a hormone, a growth factor or a cytokine.
 36. A method of making expanded cartilage tissue, the method comprising culturing a plurality of cartilage tissue pieces on a solid support, wherein each cartilage tissue piece has an initial volume of less than 1 mm³, for a time and under conditions sufficient for each tissue piece to attain an expanded volume of at least 1 mm³.
 37. The expanded cartilage tissue made by the method of claim
 21. 